Flower color is an important horticultural trait and is mainly produced by the flavonoid pigments, anthocyanins. Primarily produced to attract pollinators, flavonoids also protect the plant and its reproductive organs from UV damage, pests and pathogen (Brouillard and Cheminat, 1988; Gronquist et al., 2001). Classical breeding methods have been extensively used to develop cultivars with flowers varying in both the color and its intensity. The recent advance of knowledge on flower coloration at the biochemical and molecular level has made it possible to achieve this by genetic engineering (Tanaka et al., 1998).
Three different classes of anthocyanidins are responsible for the primary shade of flower color in many angiosperms: pelargonidin (orange to brick red), cyanidin (red to pink), and delphinidin (purple to blue). The anthocyanidin biosynthetic pathway is well established and most of the enzymes involved in the synthesis have been identified (FIG. 1) (Holton and Comish, 1995; Winkel-Shirley, 2001). It starts with the condensation of 4-coumaroyl-CoA and malonyl-CoA by chalcone synthase (CHS) to produce tetrahydroxychalcone. This is converted to a dihydroflavonol by the sequential action of chalcone isomerase (CHI) and flavanone 3-hydroxylase (F3H). The first produced dihydroflavonol is dihydrokaempferol (DHK). DHK is then converted either to kaempferol by flavonol synthase (FLS) or to leucopelargonidin by dihydroflavonol 4-reductase (DFR). Alternatively, DHK can be hydroxylated to dihydroquercetin (DHQ) or dihydromyricetin (DHM) by flavonoid 3′-hydroxylase (F3′H) or flavonoid 3′5′-hydroxylase (F3′5′H). DHQ and DHM are further converted to their respective flavonols (quercetin and myricetin) by FLS or may be reduced by DFR to yield leucoanthocyanidins (leucocyanidin and leucodelphinidin). The anthocyanidins that are synthesized from the leucoanthocyanidins by anthocyanidin synthase (ANS) are then glycosylated by flavonoid 3-O-glucosyl transferase (3GT) to produce anthocyanins. Further modification by rhamnosylation, methylation, or acylation results in a wide variety of anthocyanins (Kroon et al., 1994; Ronchi et al., 1995; Fujiwara et al., 1997; Yoshida et al., 2000; Yabuya et al., 2001). The spectral difference in flower color is mainly determined by the ratio of different classes of anthocyanins and other factors such as vacuolar pH, co-pigmentation, metal ion complexation and molecular stacking (Holton et al., 1993; Markham and Ofman, 1993; Mol et al., 1998; Tanaka et al., 1998; Aida et al., 2000). The final shade may be altered further by various factors including the shape of the epidermal cells or the presence of starch that gives creaminess (Markham and Ofman, 1993; Noda et al., 1994; Mol et al., 1998; van Houwelingen et al., 1998).
Genetic engineering to alter flower color has been attempted using various genes. Some species lack a particular color due to the absence of a biosynthetic gene or the substrate specificity of an enzyme in the pathway. For example, carnation lacks blue/purple colored flowers due to the absence of F3′5′H, while petunia lacks orange and brick-red flowers due to the inability of its DFR to reduce DHK (Gerats et al., 1982; Forkmann and Ruhnau, 1987). Genetic engineering of blue/purple colored carnation was achieved by introducing petunia F3′5′H gene and orange-colored petunia was developed by introducing DFR from other species (Meyer et al., 1987; Brugliera et al., 2000; Johnson et al., 2001). The modulation of color intensity has been another target for genetic engineering. Expression of biosynthetic genes such as CHS, F3H, and DFR in sense or antisense directions has been the most exploited method (van der Krol et al., 1990; Courtney-Gutterson et al., 1994; Jorgensen et al., 1996; Tanaka et al., 1998). The resulting sense suppression or antisense inhibition is collectively called post-transcriptional gene silencing (PTGS). Though these approaches have been fairly successful in the down-regulation of pigment synthesis, the necessity of cloning the gene of interest from a specific species or closely related species is the major drawback. Further, it is difficult to limit the PTGS to specific tissues (Palauqui et al., 1997; Voinnet and Baulcombe, 1997; Voinnet et al., 1998; Fagard and Vaucheret, 2000; Crete et al., 2001; Vaucheret et al., 2001). Alternatively, transcription factors that can either activate or repress the transcription of anthocyanin biosynthetic genes have been shown to be useful in regulating color intensity in model plants such as Arabidopsis, tobacco, and Petunia (Lloyd et al., 1992; Mol et al., 1998; Borevitz et al., 2000; Aharoni et al., 2001). The overexpression of transcription factors, however, generally alters the expression of many genes, thus the commercial viability of such transgenic flowers has yet to be determined (Lloyd et al., 1994; Bruce et al., 2000).
The biochemical and structural characterization of CHS suggests the possibility of designing a dominant-negative CHS that can be used to regulate flower color intensity. CHS is the first enzyme in the synthesis of various flavonoids including anthocyanins. It functions as a homodimer and carries out a series of reactions at a single active site (Tropf et al., 1995). The enzyme condenses a molecule of 4-coumaroyl-CoA with three of malonyl-CoA and folds the tetraketide intermediate into an aromatic ring structure (Schroder, 1999). Site-directed mutagenesis and inhibitor studies have identified the conserved cysteine and histidine residues that are important for the catalytic function of CHS (Lanz et al., 1991; Suh et al., 2000). The crystal structure of alfalfa CHS indicates that the conserved Cys164, Phe215, His303 and Asn336 form the catalytic active site (Ferrer et al., 1999). In addition, the crystal structure also shows that Met137 from the adjoining monomer extends into the cyclization pocket of CHS. This suggests that a CHS monomer requires the methionine from the adjoining monomer for its activity. Based on this structural information, we have generated CHS that has alanine instead of cysteine at the active site and either glycine or lysine instead of the methionine. The mutation of cysteine to alanine will result in the inactive form of CHS while the mutation of methionine to glycine or lysine will inactivate the function of an adjoining CHS if the methionine is really important as suggested by the crystal structure. Using transgenic Arabidopsis, we demonstrate that the mutated CHS is indeed dominant-negative. Our results confirm the importance of the methionine residue and demonstrate the utility of the dominant-negative CHS in modulating flower colour intensity even in a distantly related species.
Accordingly, the object of this invention is to provide modified Mazus CHS, which encodes a modified chalcone synthase that has alanine instead of cysteine at the 165th amino acid of Mazus CHS and either glycine or lysine instead of methionine at the 138th amino acid of Mazus CHS.
It is also an object herein to provide transgenic plants expressing the modified Mazus CHS that confers a phenotype characterized by the decreased content of anthocyanins in the plants.